Delivery of therapeutic agents by a collagen binding protein

ABSTRACT

Methods of delivering therapeutic agents by administering compositions including a bacterial collagen-binding polypeptide segment linked to the therapeutic agent to subjects in need of treatment with the therapeutic agent are provided. Methods of treating hyperparathyroidism, and hair loss using compositions comprising a collagen binding polypeptide and a PTH/PTHrP receptor agonist are provided. In addition, methods of reducing hair regrowth by administering a composition including a collagen binding polypeptide and a PTH/PTHrP receptor antagonist are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a national stage filing under 35 U.S.C. ofInternational Application No. PCT/US2013/025541, filed Feb. 11, 2013,which claims the benefit of priority of U.S. Provisional PatentApplication No. 61/596,869, filed Feb. 9, 2012, and of InternationalApplication No. PCT/US2012/069831, filed Dec. 14, 2012, all of which areincorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with United States government support awarded bythe National Institutes of Health grant number NCRR COBRE 8P30GM103450and INBRE GM103429. The United States may have certain rights in thisinvention.

SEQUENCE LISTING

A Sequence Listing accompanies this application and is incorporatedherein by reference in its entirety. The Sequence Listing was filed withthe application as a text file on Feb. 11, 2013.

INTRODUCTION

Delivery of therapeutic agents to sites within the body of a subjectwhere a particular therapeutic agent is needed in order to be effectiveis a developing area. Such delivery systems will allow more efficientuse of therapeutic agents while reducing toxicity caused by sometherapeutic agents. Use of targeted liposomes or polypeptides, such asantibodies, to target therapeutic agents to particular sites within thebody has proved successful, but additional delivery agents are needed.

Alopecia (hair loss) is a psychologically and emotionally distressingevent with multiple causes. Alopecia occurs most commonly inmale-pattern baldness, affecting approximately two thirds of males byage 35; a similar pattern of hair loss can be observed in females withpolycystic ovarian syndrome. In both of these disorders, the hair lossis androgen mediated. Alopecia can also occur as an autoimmune disease,termed alopecia areata; a disorder which affects 1.7% of the population.It can occur as a side-effect of medical treatments, particularly inchemotherapy, with 65-85% of chemotherapy patients experiencing somedegree of alopecia. Psychological consequences of hair loss have beenwell studied in the chemotherapy setting. Chemotherapy-induced alopecia(CIA) can result in anxiety, depression, a negative body image, loweredself-esteem and a reduced sense of well-being. In fact, 47-58% of femalecancer patients consider hair loss to be the most traumatic aspect ofchemotherapy, and 8% would decline treatment for fear of hair loss. Inaddition to these studies in chemotherapy patients, evidence exists inother forms of alopecia to support therapy to reduce psychologicalconsequences of hair loss. Thus a new treatment to stop hair loss orspeed hair regrowth would be beneficial.

While drugs with mild anti-androgenic effects (i.e. spironolactone) hadbeen used with limited success as therapy for alopecia, the firsteffective medication for alopecia was minoxidil (Rogaine). Thisantihypertensive has an observed side-effect of causing hair growth, andis now used as topical therapy for many forms of alopecia. However,responses are incomplete, with some subjects showing only slowing ofhair loss rather than actual regrowth. Finasteride (Propecia) is a neweragent that blocks conversion of testosterone to dihydrotestosterone,resulting in improvements in androgenic alopecia at the expense ofpartial systemic androgen blockade. However, response rates withlong-term (10 years) therapy are only around 50%. Overall, despiteconsiderable research in this area, there is still no adequate therapyfor hair loss.

In addition, unwanted hair growth is a cosmetic issue many people dealwith on a regular basis. Unwanted hair growth on the face, legs, arms,chest or back is a growing cosmetic problem. Many people use lasertherapy, waxing or other therapies to remove unwanted hair. There arecurrently no topical pharmaceuticals to limit hair growth.

SUMMARY

Provided herein are methods of delivering therapeutic agents byadministering compositions including a bacterial collagen-bindingpolypeptide segment linked to the therapeutic agent to subjects in needof treatment with the therapeutic agent.

In one aspect, methods of treating hyperparathyroidism by administeringa composition comprising a bacterial collagen-binding polypeptidesegment linked to a PTH/PTHrP receptor agonist to a subject areprovided.

In a further aspect, methods of slowing hair growth or regrowth afterremoval by administering a composition comprising a bacterialcollagen-binding polypeptide segment linked to a PTH/PTHrP receptorantagonist to a subject are provided.

In a still further aspect, methods of increasing hair growth or thespeed of hair re-growth after removal or loss by administering acomposition comprising a bacterial collagen-binding polypeptide segmentlinked to a PTH/PTHrP receptor agonist to a subject are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sequence alignment showing the alignment of several M9Bbacterial collagenases from the Bacillus and Clostridium families. Thesequences are also provided in the Sequence Listing, filed herewith asfollows: ColG s3b (SEQ ID NO: 13); Csporogenes C (SEQ ID NO: 14):CbotulinumA3C (SEQ ID NO: 15); Bcereus_AH603 (SEQ ID NO: 16); Banthracis(SEQ ID NO: 17); BB14905 B (SEQ ID NO: 18): Lsphaericus B (SEQ ID NO:19); CcereusG9842 (SEQ ID NO: 20); Bmycoides (SEQ ID NO: 21);Bweihensteph (SEQ ID NO: 22): Bbrevis A (SEQ ID NO: 23); Bbrevis B (SEQID NO: 24): CperfringensB (SEQ ID NO: 25); Csporogenes B (SEQ ID NO:26): CbotulinumA3B (SEQ ID NO: 27); Csporogenes A (SEQ ID NO: 28);CbotulinumA3A (SEQ ID NO: 29): Csordellii B (SEQ ID NO: 30); ColG s3a(SEQ ID NO: 31) CperfringensA (SEQ ID NO: 32) Csordellii A (SEQ ID NO:33) and ColH s3 (SEQ ID NO: 34). The residues shown in blue areimportant for collagen binding activity, those shown in green areimportant for maintaining the architecture or protein folding. Both ofthese are also underlined for the top and bottom sequences. Residuesshown in red are critical for Ca²⁺ binding and those in orange arecritical for positioning the Ca²⁺ binding residues.

FIG. 2 shows the tissue distribution of S³⁵-PTH-CBD 1 hour and 12 hoursafter subcutaneous injection. Note the skin outline.

FIG. 3 is a set of photographs documenting the hair growth on the backof mice at day 36 after depilation, treatment groups as indicated(Antagonist PTH(7-33)-CBD, Agonist PTH-CBD).

FIG. 4 is a set of photographs showing the histology at Day 36 after theindicated treatment. Skin samples were taken from the dorsal region andprocessed for Hematoxylin and Eosin (H&E) staining. Representativesections are shown from each treatment group as indicated.(Antagonist=PTH(7-33)-CBD, Agonist=PTH-CBD).

FIG. 5 is a graph showing the hair follicle counts per high poweredfield. Anagen VI hair follicles were counted by two independentobservers in a blinded fashion. Results are expressed as mean+/−standarddeviation. **=p<0.01 vs. no chemo ANOVA followed by Dunnett's test.(Antagonist=PTH(7-33)-CBD, Agonist PTH-CBD).

FIG. 6 is a set of photographs showing the hair growth on the back ofthe mice after each of the indicated treatments and a graph showing theresults of a grey scale analysis of the hair at the injection site overtime after the injection.

FIG. 7 is a set of photographs showing the hair on the back of miceafter the indicated treatment without prior depilation.

FIG. 8 is a set of photographs and a graph showing the grey scaleanalysis of hair growth on the backs of mice comparing the indicatedtreatments with the PTH-CBD being administered prior to the chemotherapyas opposed to after chemotherapy began.

FIG. 9 is a photograph of three mice 13 days after waxing to remove hairand treatment with PTH-CBD, PTH antagonist-CBD or vehicle alone.

FIG. 10 is a set of photographs of mice showing hair regrowth in a modelof alopecia areata after treatment with a control or with PTH-CBD.

FIG. 11 is a graph showing the endogenous parathyroid hormone levels inovarectomized aged rats injected with a single dose of human PTH-CBD 6months prior to sacrifice.

DETAILED DESCRIPTION

The effects of PTH agonists and antagonists on hair growth have beenstudied for over almost 15 years. PTH has a common receptor withPTH-related peptide (PTHrP), which is normally produced by dermalfibroblasts. PTHrP affects keratinocyte proliferation/differentiationand modulates the hair cycle. Most of the testing on hair growth effectshas been performed with PTH antagonists, as indications from initialtesting were that these were the most effective agents. Both injectedand topical formulations have been tested in animal models ofchemotherapy-induced alopecia and in the SKH-1 hairless mouse. Part ofthe effect of PTH antagonists on hair growth is to transition the hairfollicles into a dystrophic catagen stage, which protects them fromchemotherapeutic damage. However, clinical trials of topical PTHantagonists for chemotherapy-induced alopecia by MI Pharmaceuticals werediscontinued in phase 2 because of limited efficacy. Thus newcompositions for treating alopecia are needed.

The problems of delivery and retention of PTH to the skin can beovercome by using collagen-targeted PTH analogs. To accomplish this, wesynthesized several fusion proteins of different PTH agonists andantagonists linked to a collagen binding domain derived from the ColH1collagenase of Clostridium histolyticum, in the studies described in theExamples, we found that the agonist compound PTH-CBD promotes transitionof hair follicles to the anagen phase and has potent effects on hairgrowth. The antagonist compound PTH(7-33)-CBD had little effect on hairgrowth in chemotherapy models and had a deleterious effect on hairregrowth after depilation. Compounds such as PTH-CBD, which promoteanagen phase transition of hair follicles, have been sought after due totheir potential to treat a large variety of disorders of hair loss.PTH-CBD appears to have a similar mechanism of action to cyclosporine,which also promotes transition of hair follicles to anagen phase,although the mechanism is less likely to be the result of direct effectson WNT signaling. While clinical use of cyclosporine for this purpose islimited by systemic toxicity, PTH-CBD has not shown toxic effects, evenwith systemic administration.

Thus in another aspect, methods of increasing hair growth are providedherein. The methods include administering a CBD linked to a PTH/PTHrPreceptor agonist to a subject in need of treatment to induce hair growthor stop hair loss. The method is applicable to individuals withalopecia, including chemotherapy induced alopecia, but also alopeciaareata, alopecia caused by male pattern baldness, polycystic ovariansyndrome or other hair loss. The compositions may be administeredlocally or topically to treat hair loss.

In another aspect, methods of slowing hair growth or regrowth after ahair removal procedure by administering a CUD linked to a PTH/PTHrPreceptor antagonist to a subject are provided. In one embodiment, thePTH antagonist composition is applied locally, topically. The PTHantagonist may be applied after a hair removal procedure to prevent orslow hair regrowth. As described in the Examples, we have demonstratedthat hair regrowth is slowed after waxing in animals treated withCBD-PTH antagonist as compared to control animals treated with PTH CBDor vehicle alone. The compositions may be administered locally ortopically to block hair growth.

Also provided herein are methods of treating hyperparathyroidism byadministering PTH-CBD to a subject in need of treatment forhyperparathyroidism. In one embodiment the PTH administered to thesubject may be a PTH from a different species. As shown in the Examplesa single administration of CBD-PTH to ovarectomized aged rats was ableto reduce the amount of endogenous PTH produced by the animal. Thus,administration of PTH-CBD to individuals suffering fromhyperparathyroidism may experience a decrease in symptoms associatedwith hyperparathyroidism and have decreased levels of PTH afteradministration of PTH-CBD.

The collagen-binding polypeptide segment and the therapeutic agent maybe chemically cross-linked to each other or may be polypeptide portionsof a fusion protein. The terms “fusion protein” and “fusion polypeptide”may be used to refer to a single polypeptide comprising two functionalsegments, e.g., a collagen-binding polypeptide segment and a polypeptidebased therapeutic agent, such as PTH/PTHrP receptor agonist polypeptidesegment. The fusion proteins may be any size, and the single polypeptideof the fusion protein may exist in a multimeric form in its functionalstate, e.g. by cysteine disulfide connection of two monomers of thesingle polypeptide. A polypeptide segment may be a synthetic polypeptideor a naturally occurring polypeptide. Such polypeptides may be a portionof a polypeptide or may comprise one or more mutations. The twopolypeptide segments of the fusion proteins can be linked directly orindirectly. For instance, the two segments may be linked directlythrough, e.g., a peptide bond or chemical cross-linking, or indirectly,through, e.g., a linker segment or linker polypeptide. The peptidelinker may be any length and may include traditional or non-traditionalamino acids. For example, the peptide linker may be 1-100 amino acidslong, suitably it is 5, 10, 15, 20, 25 or more amino acids long suchthat the collagen binding portion of the fusion polypeptide can mediatecollagen binding and the therapeutic agent can have its therapeuticeffect. Peptide linkers may include but are not limited to a PKD(polycystic kidney disease) domain from a collagenase or other proteinsuch as in SEQ ID NO: 2, a GST or His-tag, or a Ser or Gly linker.

The collagen-binding polypeptide segment is a polypeptide that bindscollagen and may be part of a larger fusion protein, bioactive agent, orpharmaceutical agent. Determination of whether a composition,polypeptide segment, fusion protein, or pharmaceutical or bioactiveagent binds collagen can be made as described in U.S. Patent PublicationNo. 2010/0129341, which is incorporated herein by reference in itsentirety. Briefly, the composition is incubated with collagen in bindingbuffer, and the mixture is then filtered through a filter that wouldotherwise allow it to pass through but that blocks the collagen andtherefore holds back materials that bind to the collagen. The filtrateis then assayed for the presence of the composition, polypeptidesegment, fusion protein, or pharmaceutical or bioactive agent. Suitably,at least 80%, 85%, 90%, 95%, 98% or more suitably at least 99% of thecollagen-binding composition, polypeptide segment, fusion protein, orpharmaceutical or bioactive agent is retained by the filter in thisassay, as compared to when the filtration is performed without collagen.

The collagen-binding polypeptide segment may be a bacterialcollagen-binding polypeptide segment. It may be a Clostridiumcollagen-binding polypeptide segment. The collagen-binding polypeptidesegment may be a segment of a collagenase, or a bacterial collagenase,or a Clostridium collagenase. Suitably the polypeptide segment is only aportion of the collagenase and the collagen-binding polypeptide segmentdoes not have collagenase activity. The collagen-binding polypeptide maybe a bacterial M9B (including those derived from Bacillus spp. andClostridium spp.) or M9A (including those derived from Vibrio spp.)collagen-binding protein or a collagen-binding peptide derived from sucha protein. By “derived from” we mean that the peptide is a fragment ofthe full-length protein, a peptide that has amino acid changes relativeto the wild-type protein or a combination thereof. The key is that thepeptide retains the ability to bind collagen. For example, a peptide maybe derived from a protein by selecting a region of the protein capableof binding to collagen. Compositions including a bacterial collagenaseas a collagen binding peptide are described in US Patent Publication No.2010/0129341, which is hereby incorporated herein by reference in itsentirety.

FIG. 1 shows a sequence alignment of the collagen-binding region ofseveral M9B bacterial collagen-binding proteins included as SEQ ID NOs:13-34. As can be seen from the sequence alignment, these proteins have arelatively small amount of sequence identity (about 30%), but they allbind to collagen in a similar fashion and are believed to have similarconformation as discussed in the Examples. Notably, the proteins shareseveral amino acids critical for binding to collagen. Thus any of thepeptides shown in FIG. 1 or collagen-binding fragments thereof can beused in the compositions and methods described herein (i.e., SEQ H) NOs:13-34 can be used interchangeably).

In FIG. 1, the amino acid residues critical for the conformation of thepeptide and for the collagen-binding activity are underlined and shownin green and blue respectively. The key amino acid residues forcollagen-binding are as follows:

a phenylalanine, histidine or tyrosine at position 959 of ColG in FIG. 1(position 63 of SEQ ID NO: 13), position 968 of the ColH sequence of SEQID NO: 6, position 928 of the ColH sequence of FIG. 1 (position 63 ofSEQ ID NO: 34) or a similar position of one of the other sequences shownin FIG. 1;

a tyrosine, serine or phenylalanine at position 970 of ColG in FIG. 1(position 74 of SEQ ID NO: 13), position 977 of the ColH sequence of SEQID NO: 6, position 937 of the ColH sequence of FIG. 1 (position 72 ofSEQ ID NO: 34) or a similar position of one of the other sequences shownin FIG. 1;

a tyrosine at position 994 of ColG in FIG. 1 (position 98 of SEQ ID NO:13), position 1002 of the ColH sequence of SEQ ID NO: 6, position 962 ofthe Corn sequence in FIG. 1 (position 97 of SEQ ID NO: 34) or a similarposition of one of the other sequences shown in FIG. 1;

a tyrosine, phenylalanine or histidine at position 996 of ColG in FIG. 1(position 100 of SEQ ID NO: 13), position 1004 of the ColH sequence ofSEQ ID NO: 6, position 964 of the ColH sequence in FIG. 1 (position 99of SEQ ID NO: 34) or a similar position of one of the other sequencesshown in FIG. 1. The critical segment for collagen binding is aminoacids 90-100 of SEQ ID NO: 13 which corresponds to amino acids 89-97 ofSEQ ID NO: 34 or the equivalent thereof from another bacterial collagenbinding protein such as the sequences provided in the alignment of FIG.1 (SEQ ID NOs: 14-33). Thus a peptide with relatively low sequenceidentity, sharing the structure and function of the ColG protein mayalso be used as a collagen binding domain (CBD) herein.

In one embodiment, the collagenase is ColH, SEQ ID NO: 6. Thecollagen-binding polypeptide segment may be or may include residues901-1021 of SEQ ID NO:6 (residues 34-158 of SEQ ID NO:1), or a fragmentof residues 34-158 of SEQ ID NO:1 at least 8, 10, 12, 15, 20, 25, 30,40, 50, 60, 70, 80, 90, 100, 110 or 120 amino acid residues in length.The collagen-binding polypeptide segment is at least 50%, 60%, 70%, 80%,or at least 85%, at least 90%, at least 95%, at least 96%, at least 98%,or at least 99% identical to residues 34-158 of SEQ ID NO: 1 or to anyone of SEQ ID NO: 13-34. The collagen-binding polypeptide segment may beor may comprise a fragment of residues 901-1021 of SEQ NO:6, e.g., afragment of at least 8, at least 10, at least 20, at least 30 at least40, or at least 50 consecutive amino acid residues of residues 901-1021of SEQ ID NO:6. Suitably the collagen-binding polypeptide consists ofresidues 894-1008, 894-1021, 901-1021, or 901-1008 of SEQ ID NO: 6 or ahomolog thereof as shown by the sequence alignment in FIG. 1. Asdescribed more fully in the examples, the entire collagen bindingpolypeptides shown in FIG. 1 are not required for collagen binding. Thecollagen binding segment may be as short as eight amino acids and mayinclude, e.g., amino acids 89-97 of SEQ ID NO: 34, 90-98 of SEQ ID NO:13 or 994-1002 of SEQ ID NO: 6. A ten amino acid collagen bindingpeptide may include e.g., amino acids 89-99 SEQ ID NO: 34, 90-100 SEQ IDNO 13 or 994-1004 SEQ ID NO 6. A 30 amino acid collagen binding segmentmay include e.g., amino acids 70-100 of SEQ ID NOs: 13 or 34 or 975-1005of SEQ ID NO: 6. A forty amino acid collagen binding peptide may includee.g, amino acids 60-100 of SEQ ID NOs: 13, 34 or 964-1004 of SEQ ID NO:6, or 59-99 of SEQ ID NO: 34. A fifty amino acid collagen bindingsegment may include e.g., amino acids 50-100 or 55-105 of SEQ ID NOs:13-34 or 9060-1010 of SEQ ID NO: 6. Similar shorter peptides derived forcorresponding sequences of SEQ ID NOs: 14-33 may also be used.

The collagen-binding polypeptide segment may be or may include residues807-1021 of SEQ ID NO: 6 (residues 37-251 of SEQ ID NO: 2), or afragment of residues 807-1021 of SEQ ID NO:6 at least 8, 10, 12, 15, 20,25, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 amino acid residues inlength. Residues 807-901 comprise the polycystic kidney disease (PKD)domain of the collagen-binding protein. Those of skill in the art willappreciate that other linkers could be used to link the collagen-bindingpeptide to a therapeutic agent as outlined above.

Among other proteins the collagen-binding segment can be derived fromare ColG (Matsushita et al., (1999) J. Bacteria 181:923-933), a class Icollagenase from Clostridium histolyticum. ColH is a class IIcollagenase (Yoshihara et al., (1994) J. Bacteriol. 176: 6489-6496). Thecollagen-binding polypeptide segment may also be a polypeptide segmentfrom any one of the protein sequences provided in FIG. 1 which alignscollagen-binding peptides from members of Clostridium and Bacillus.Those of skill in the art will appreciate that other members of thiscollagen-binding protein family may be useful in the methods describedherein.

The PTH/PTHrP receptor agonist polypeptide segment may be a syntheticpolypeptide or a naturally occurring polypeptide. Such polypeptides maybe a portion of a polypeptide or may comprise one or more mutations. Themutations may make the PTH/PTHrP receptor agonist a better or worseagonist as compared to the wild-type PTH/PTHrP. Agonist activity withthe PTH/PTHrP receptor can be assayed as described in Example 3 below bya cAMP stimulation assay. An agonist will stimulate cAMP synthesis inthe assay described. Suitably, an agonist can activate receptor activityat least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or oven 110%or 120% as much as wild-type PTH(1-34).

The PTH/PTHrP receptor agonist polypeptide segment is a PTH or PTHrPpolypeptide segment. One human isoform of PTH is SEQ ID NO: 7. One humanisoform of PTHrP is SEQ ID NO:8. While the human isoforms are provided,those of skill in the art will appreciate that other non-human-derivedisoforms may be used as well. Such non-human-derived isoforms may beable to interact with human PTH/PTHrP receptor and vice versa. ThePTH/PTHrP receptor agonist polypeptide segment may be or may includeresidues 1-33 of SEQ ID NO:1 (residues 1-33 of PTH (SEQ ID NO: 7)). ThePTH/PTHrP receptor agonist polypeptide segment may be or may includeresidues 1-34 of PTH (SEQ ID NO: 7). In other embodiments, it is afragment of residues 1-34 of PTH (SEQ ID NO: 7). In other embodiments,the PTH/PTHrP receptor agonist polypeptide segment may be or may includeresidues 1-84 of PTH (SEQ ID NO:7). In other embodiments, the PTH/PTHrPreceptor agonist polypeptide segment may be or may include residues 1-14of PTH (SEQ ID NO 7) or residues 1-7 of PTH (SEQ ID NO: 7). The keyamino acids for binding to the PTH receptor ad an agonist are aminoacids 1, 2 and 5 of PTH. In still other embodiments, the PTH/PTHrPreceptor agonist is a PTH or PTHrP polypeptide segment for any otherspecies.

The PTH/PTHrP receptor antagonist can include in one embodimentPTH(7-34), i.e., residues 7-34 of PTH (SEQ ID NO: 7). In anotherembodiment, it is or includes residues 7-33 of PIE (SEQ ID NO: 7). Inother embodiments, it is a fragment of residues 7-34 of SEQ ID NO: 8. Inanother embodiment, the PTH/PTHrP receptor antagonist includes PIE(7-14), residues 7-14 of PTH (SEQ ID NO: 7). In another embodiment, thePTH/PTHrP receptor antagonists include ((−1)-33) of PTH/PTHrP. Inanother embodiment, the PTH/PTHrP receptor antagonists include residues1-14 of PTH with an N-terminal extension. Adding an N-terminal extensionto PTH or active N-terminal fragments of PTH converts the PTH peptidesto antagonists. The N-terminal extension can be 1, 2, 3, 4, 5, or moreamino acids in length. The identity of the amino acids in the N-terminalextension is typically not important. In one embodiment, the PTH/PTHrPreceptor antagonist includes residues 1-33 of PTH with a Gly-Serextension at the N-terminus (SEQ ID NO:11). In another embodiment, thePTH/PTHrP receptor antagonist includes PTHrP(7-34), i.e., residues 7-34of SEQ ID NO:8, or a fragment of residues 7-34 of SEQ NO:8. In anotherembodiment, the PTH/PTHrP receptor antagonist includes mouse TIP(7-39)(See Hoare S R, Usdin T B. 2002. Specificity and stability of a new PTH1receptor antagonist, mouse TIP(7-39). Peptides 23:989-98.). OtherPTH/PTHrP receptor antagonists that may be used in the fusion proteinsare also disclosed in Hoare et al. The PTH/PTHrP receptor antagonist maybe a fragment of at least 8, 10, 12 or more amino acids from residues1-34 of SEQ ID NO:7. In other embodiments the PTH/PTHrP receptorantagonist may be PTH/PTHrP receptor antagonist polypeptide from anotherspecies.

In one embodiment, the therapeutic agent or PTH/PTHrP receptor agonistor antagonist polypeptide segment is N terminal to the collagen-bindingpolypeptide segment in the fusion protein. That is, the two polypeptidesegments each have an N-terminal and a C-terminal, and the N-terminal ofthe collagen-binding polypeptide segment is linked directly orindirectly, e.g., through a linker polypeptide segment (such as PKD, aGlycine or Serine linker) to the C-terminal of the therapeutic agent orPTH/PTHrP agonist or antagonist polypeptide segment.

The fusion proteins described above comprising (a) a collagen-bindingpolypeptide segment linked to (b) a therapeutic agent or a PTH/PTHrPreceptor agonist or antagonist polypeptide segment can be replaced bypharmaceutical agents comprising (a) a collagen-binding polypeptidesegment linked to (b) a therapeutic agent or PTH/PTHrP receptor agonistor a non-peptidyl PTH/PTHrP receptor agonist. An example of anon-peptidyl PTH/PTHrP receptor agonist is compound AH3960 (Rickard etal., (2007) Bone 39:1361-1372).

AH13960 contains two amino groups. Amino groups in small chemicalmolecules such as AH3960 can be used to cross-link the therapeutic agentto amino groups on the collagen-binding polypeptide segment through across-linker such as DSG (disuccinimidyi glutarate) or through thecombination of SANK (succinimidyl-4-hydrazinonicotinate acetonehydrazone) and SFB (succinimidyl-4-formyl benzoate). Therapeutic agentscan be cross-linked through their amino group to a carboxyl group of thecollagen-binding polypeptide segment by EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) or viceversa. These cross-linking products are available from Pierce(piercenet.com, Thermo Fisher Scientific Inc., Rockford, Ill.).Protocols and reaction conditions are also available in the productliterature from Pierce (piercenet.com).

In another embodiment of the pharmaceutical agents comprising (a) acollagen-binding polypeptide segment; linked to (b) a polypeptidetherapeutic agent or a PTH/PTHrP receptor agonist or antagonistpolypeptide segment, segment (a) and segment (b) are separatepolypeptides, and the two polypeptides are linked by chemicalcross-linking. The two polypeptides can be cross-linked through aminogroups by reagents including DSG (disuccinimidyl glutarate) orglutaraldehyde. They can also be cross-linked through amino groups byderivatizing one polypeptide with SANH(succinimidyl-4-hydrazinonicotinate acetone hydrazone) and the otherwith SFB (succinimidyl-4-formyl benzoate), and then mixing the twoderivatized polypeptides to cross-link. The two polypeptides can becross-linked between an amino group of one polypeptide and a carboxyl ofthe other by reaction with EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride). Thepolypeptides can also be cross-linked (e.g., covalently coupled) by anyother suitable method known to a person of ordinary skill in the art.These cross-linking reagents are available from Pierce (piercenet.com,Thermo Fisher Scientific Inc., Rockford, Ill.). Protocols and reactionconditions are also available in the product literature from Pierce(piercenet.com). These and other applicable cross-linking methods aredescribed in U.S. published patent applications 2006/0258569 and2007/0224119.

The compositions described herein may be administered by any means knownto those skilled in the art, including, but not limited to, oral,topical, intranasal, intraperitoneal, parenteral, intravenous,intramuscular, intradermal or subcutaneous. Thus the compositions may beformulated as an ingestable, injectable, topical or suppositoryformulation. The composition may be formulated for administration byinjection to result in systemic administration or local administration.The compositions may also be delivered with in a liposomal ortime-release vehicle. The compositions may also be delivered in asite-directed delivery vehicle, such as but not limited to, a targetedliposome or an absorbable collagen sponge carrier or other implant.

The inventors have found that when administering compositions includinga CBD subcutaneously it binds locally at the site of injection if thecomposition is dissolved in neutral pH buffer. But if the composition isdissolved in a low pH buffer, for example a buffer having pH 5.0 or pH4.5 or below, the collagen-binding domain does not bind collagen, andthe composition has time to disperse systemically before it bindscollagen elsewhere in the body at neutral pH. Thus systemicadministration of the compositions involves administering thecomposition dissolved in buffer or aqueous solution at a pH lower thanabout 5.0 or at pH 4.5 or below. In another embodiment, systemicadministration of the compositions involves administering the fusionproteins dissolved in aqueous solution at lower than about 6.0.Alternatively, if the skin condition is localized, the compositionsdescribed herein may be administered in a buffer with a pH of 6.0, 6.5,7.0, 7.5 or above in order to allow for localized delivery of thecompositions to the affected area of the skin.

Pharmaceutical compositions for topical administration may also beformulated using methods and compositions such as those available tothose skilled in the art. For example, gels, creams or liposomepreparations may be suitable for topical delivery. These deliveryvehicles may be formulated to mediate delivery to the lower layers ofthe skin or to allow for extended release of the pharmaceutical at thesite of application.

The compositions can be administered as a single dose or as divideddoses. For example, the composition may be administered two or moretimes separated by 4 hours, 6 hours, 8 hours, 12 hours, a day, two days,three days, four days, one week, two weeks, or by three or more weeks.Optionally, such treatment may be repeated, for example, every 1, 2, 3,4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 months. The composition is expected tobe more effective than a comparable or control composition comprisingthe therapeutic agent or a PTH/PTHrP receptor agonist that is not linkedto a collagen-binding protein. In one embodiment, a smaller amount ofthe composition may be used or the composition may be administered lessfrequently than a comparable composition comprising the therapeuticagent or a PTH/PTHrP receptor agonist which is not linked to acollagen-binding protein.

The dosage amounts and frequencies of administration provided herein areencompassed by the terms therapeutically effective and prophylacticallyeffective. The individual doses of pharmaceutical agents comprising acollagen-binding polypeptide segment linked to a therapeutic agent maybe approximately the same on a molar basis as doses used for thetherapeutic agent alone. It is expected that the pharmaceutical agentscomprising a collagen-binding polypeptide segment linked to atherapeutic agent may be administered less frequently, because linkingthe agent to the collagen-binding polypeptide segment gives it much moreprolonged activity in vivo.

Administration of the compositions to a subject in accordance with theinvention appears to exhibit beneficial effects in a dose-dependentmanner. Thus, within broad limits, administration of larger quantitiesof the compositions is expected to achieve increased beneficialbiological effects than administration of a smaller amount. Moreover,efficacy is also contemplated at dosages below the level at whichtoxicity is seen.

It will be appreciated that the specific dosage administered in anygiven case will be adjusted in accordance with the compositions beingadministered, the disease to be treated or inhibited, the condition ofthe subject, and other relevant medical factors that may modify theactivity of the agent or the response of the subject, as is well knownby those skilled in the art. For example, the specific dose for aparticular subject depends on age, body weight, general state of health,diet, the timing and mode of administration, the rate of excretion,medicaments used in combination and the severity of the particulardisorder to which the therapy is applied. Dosages for a given patientcan be determined using conventional considerations, e.g., by customarycomparison of the differential activities of the compositions of theinvention and of the therapeutic agent administered alone, such as bymeans of an appropriate conventional pharmacological or prophylacticprotocol.

The maximal dosage for a subject is the highest dosage that does notcause undesirable or intolerable side effects. The number of variablesin regard to an individual prophylactic or treatment regimen is large,and a considerable range of doses is expected. The route ofadministration will also impact the dosage requirements. It isanticipated that dosages of the compositions will reduce symptoms of thecondition being treated by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or 100% compared to pre-treatment symptoms or symptoms is leftuntreated. It is specifically contemplated that pharmaceuticalpreparations and compositions may palliate or alleviate symptoms of thedisease without providing a cure, or, in some embodiments, may be usedto cure the disease or disorder.

Suitable effective dosage amounts for administering the compositions maybe determined by those of skill in the art, but typically range fromabout 1 microgram to about 10,000 micrograms per kilogram of body weightweekly, although they are typically about 1,000 micrograms or less perkilogram of body weight weekly. In some embodiments, the effectivedosage amount ranges from about 10 to about 10,000 micrograms perkilogram of body weight weekly. In another embodiment, the effectivedosage amount ranges from about 50 to about 5,000 micrograms perkilogram of body weight weekly. In another embodiment, the effectivedosage amount ranges from about 75 to about 1,000 micrograms perkilogram of body weight weekly. The effective dosage amounts describedherein refer to total amounts administered, that is, if more than onecompound is administered, the effective dosage amounts correspond to thetotal amount administered.

The effectiveness of the compositions described herein may be enhancedby at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 100% relative to acontrol treated with the therapeutic agent alone. It will be appreciatedthat the effectiveness of the treatment in any given case will beenhanced variably in accordance with the specific compositions used, thetype of disease being treated, the condition of the subject, thespecific formulations of the compounds and other relevant medicalfactors that may modify the activity of the compositions or theresponses of the subject as is appreciated by those of skill in the art.

The following examples are meant only to be illustrative and are notmeant as limitations on the scope of the invention or of the appendedclaims. All references cited herein are hereby incorporated by referencein their entireties.

EXAMPLES Example 1 Structural Comparison of ColH and ColGCollagen-Binding Domains

The C-terminal collagen-binding domain (CBD) of collagenase is requiredfor insoluble collagen fibril binding and for subsequent collagenolysis.The high resolution crystal structures of ColG-CBD (s3b) and ColH-CBD(s3) the molecules resemble one another closely (r.m.s.d. C_(α)=1.5 Å),despite sharing only 30% sequence identity. Five out of six residueschelating Ca²⁺ are conserved. The dual Ca²⁺ binding sites in s3 arecompleted by a functionally equivalent aspartate. The three mostcritical residues for collagen interaction in s3b are conserved in s3.The general shape of the binding pocket is retained by altered loopstructures and side-chain positions. Small angle X-ray scattering datarevealed that s3 also binds asymmetrically to mini-collagen. Besides thecalcium-binding sites and the collagen-binding pocket, architecturallyimportant hydrophobic residues and hydrogen-bonding network around thecis-peptide bond are well-conserved in metallopeptidase subfamily M9B.

Common structural features described above and in Bauer et (2012) JBacteriol November 9 (which is incorporated herein by reference in itsentirety), enabled us to update the sequence alignment of the CBD in theM9B subfamily (FIG. 1). Conserved residues are important for one of fourreasons: calcium chelation (red), cis-trans isomerization of the linker(yellow), collagen-binding (blue) or protein folding (green). FIG. 1also indicates the strands of the structure along the top of the figure.

The dual calcium-binding site is formed by four chelating residues(Glu899, Glu901, Asn903, and Asp904) within the N-terminal linker, twochelating residues (Asp927 and Asp930) from the β-strand C and invariantTyr1002 hydrogen-bonds and orients Asp930. Residue numbers used in thisparagraph are of s3b. Likewise other supporting cast such as Gly921 isconserved in the middle of β-strand strategically placed to make roomfor Glu899. The dual calcium chelation site is fashioned sometimes byfunctionally equivalent residues. As mentioned, Asp897 of s3 actsequivalently to Asp927 of s3b. Asp897 equivalents are tentativelyidentified in B. brevis s3a and s3b, C. botulinum A3 s3a and C.histolyticum ColG s3a. Tridentate and divalent Asp and Glu residues areconserved with only C. sordellii s3a as the exception. The monodentateAsp904 residue is sometimes substituted by Asn. For those substituted,the net charge of the dual calcium site is neutral rather than −1.

The peptide between residues 901-902 has cis conformation in the holostate for both s3b and s3. The position 902 in other CBD molecules isPro, Asp or Asn. Pro frequently succeeds the peptide bond to easetrans-cis isomerization. The s3 molecule has Pro. In s3b, OD of Asn902hydrogen-bonds with the main-chain N of Asp904. The hydrogen-bond iscritical for the peptide isomerization. Spiriti and van der Vaart.(2010) Biochemistry 49:5314-5320, which is incorporated herein byreference in its entirety. For the remainder of CBD molecules with Aspat the position, OD of Asp could play the same role as that of Asn902.Other hydrogen-bonds identified by simulation studies important instabilizing the transition states are well conserved. Thesedonor-acceptor pairs in s3 and s3b are tabulated (Table 1). Calcium ionscould catalyze the isomerization in all the CBD molecules and theirtransition states and catalytic mechanism may look very similar.

TABLE 1 Hydrogen-bonds important in trans-cis peptide isomerization ins3b and their counterparts in s3. Important H-bonds in s3b fortransition state formation Corresponding H-bonds in s3 T910_OG1 . . .N903_NH2 S879_OG1 . . . N872_ND2 T910_OG1 . . . N900_N S879_OG1 . . .K86_N E899_OE1 . . . N903_ND2 E868_OE1 . . . N872_ND2 E899_OE2 . . .S922_N E868_OE2 . . . T891_N N902_OD1 . . . D904_N NA (N902 replacedwith P871) D930_OD2 . . . Y1002_OH D939_OD2 . . . Y97_OH Y1002_OH . . .Y932_OH NA (Y932 replaced with F901)

Non-functional residues that are important in either folding orarchitectural stability are conserved. Hydrophobic residues packedbetween the β-sheets are better conserved if they are located in thevicinity of functionally critical residues. For example, invariantTrp956 of strand E is packed between the β-sheets. The residues flanking(Thr955 & Thr957) interact with mini-collagen. Tyr932 is packed betweenthe sheets and helps positioning Tyr1002. Residues at tight turns areconserved as well. Gly975 is well conserved to allow a type II′ turn ins3b, Gly942 (Gly975 equivalent) in s3 allows Asp941 side-chain tostabilize the reverse turn. A highly conserved six-residue stretch,between residues 986 and 991, adopts a tight turn and precedes thefunctionally important strand H. The region is well ordered in thecrystal structures with low B-factors, and is the least dynamic based onNMR and limited proteolysis MALDI-TOF MS. Philominathan, et al. (2009) JBiol Chem 284:10868-10876 and Sides et al. J Am Soc Mass Spectrom.(2012) 23(3):505-19 both of which are incorporated herein by referencein their entireties. The main-chain carbonyl and amino groups of Arg985hydrogen-bond with OH of Tyr989 to stabilize the turn. Only Gly987 canmake room for the bulky Tyr989 side chain. Try990 packs against theinvariant Ala909 and conserved 3₁₀ helix.Ala909 is at the base of thelinker that undergoes α-helix→β-strand transformation. The tight turnmay ensure that collagen interacting Leu992, Tyr994, and Tyr996 would becorrectly positioned. Tyr994 is the most critical residue in interactingwith collagenous peptides. Wilson, et al. (2003) EMBO J. 22:1743-1752.The strands adjacent to strand H, i.e. strands C and E, are very wellconserved. The three antiparallel strands mold the collagen-bindingpocket. Strand F staples the β-sheets by interacting with both sheets.The β-strand first interacts in an antiparallel orientation with strandE then breaks its direction at Gly971 to interact with strand G. Inplace of Gly971, Ala or Pro is found at the location where the strandswitches its allegiance. The dual interaction of the strand helpspositioning Tyr970 to interact with mini-collagen.

Three residues shown to interact strongly with mini-collagen areconserved. The invariant Tyr994 and well conserved Tyr970 and Tyr996constitute the “hot spot”. Y994A mutation lost binding capability. SinceY994F resulted in 12-fold reduction in binding to mini-collagen, thehydroxyl group of Tyr994 may interact with collagen through ahydrogen-bond. Tyr996, which is a critical residue in bindingmini-collagen, is not so well conserved. Y996A caused 40-fold reductionin binding to the mini-collagen. Y996 is s3b is replaced with Phe in s3,though both side chains have identical orientation. In other CBDmolecules, an aromatic residue, such as Phe or His, is sometimes foundat the site. Y970A results in 12-fold reduction in binding tomini-collagen. Thr957 was found to interact with mini-collagen by¹⁵N-HSQC-NMR titration. The β-branched amino acid residues or Leu arefound at the positions equivalent to Thr957 in most of the CBDs. Sixother residues were identified by ¹⁵N-HSQC-NMR titration to interactwith mini-collagen are not very well conserved. Since divergent CBDs (s3and s3b) adopted a similar saddle-shaped binding pocket, other CBDs mayalso adopt similar collagen-binding strategy.

Divergent CBD could target different collagen sequences and couldpossibly target different collagen types; however, this structural studysuggest otherwise. Rather, all the CBD domains may bind similarly to anunder-twisted region such as the C-terminus of a collagen fibril. TheC-terminus of type I collagen is exposed in the fibril surface based onX-ray fiber diffraction experiments, and it is the most accessible sitefor the bacterial collagenase to initiate assaults. However CBD bindingonly at the C-terminal region of tropocollagen is unfounded. Goldparticle-labeled tandem ColG-CBD (s3a-s3b) labeled with gold particlebound to type I collagen fibrils exhibited no periodicity. In thecollagen fibrils, the molecules are staggered from each other by about67 nm. Therefore CBD could target partially under-twisted regions in themiddle of a tropocollagen that are also vulnerable for assaults.

Much like s3b, s3 is both compact, and extremely stable in the presenceof physiological Ca²⁺. Thus, the enzyme could degrade extracellularmatrix for prolonged time. The linker that induced structuraltransformation is a common feature found in M9B collagenase. It couldact as Ca²⁺ to trigger domain rearrangement as means of enzymeactivation. Ca²⁺ concentration in extracellular matrix is higher thanthat inside a bacterium. Both s3 and s3b bind similarly to amini-collagen, thus M9B collagenase molecules could initiatecollagenolysis from analogous structural features in various collagenfibril. Fusion protein of any CBD derived from M9B collagenase and agrowth factor should result in comparable clinical outcome.

Example 2 CBD-PTH Agonist Spurs Hair Growth and CBD-PTH AntagonistInhibits Hair Growth

In-Vitro Characterization of CBD-Linked PTH Compounds:

Collagen binding of each peptide was verified in flow-through collagenbinding assays as previously described in U.S. Patent Publication No.2010/0129341, which is incorporated herein by reference in its entirety.PTH-CBD, consisting of the first 33 amino acids of PTH linked directlyto the collagen binding domain (SEQ ID NO 1), was the most potentagonist, having a similar effect to that of PTH(1-34) (SEQ ID NO: 7) oncAMP accumulation. Ponnapakkam et al. (2011) Calcif 88:511-520. Epub2011 April 2022. Among the antagonists, PTH(7-33)-CBD (SEQ ID NO: 10)had the best combination of low intrinsic activity and high receptorblockade (not shown), similar to those seen in other PTH antagonists,including those used in hair growth studies. Peters, et al. (2001) JInvest Dermatol 117:173-178.

In Vivo Distribution of PTH-CBD:

Tissue distribution was assessed by administering ³⁵S-labelled PTH-CBDvia subcutaneous injection, followed by whole mount frozen andwhole-body autoradiography. PTH-CBD with a phosphorylation site betweenPTH(1-33) and the CBD was purified, activated and labeled with[gamma-35] ATP as described previously. Tamai et al. (2003) InfectImmun. 71:5371-5375. Approximately 10.8 meg of ³⁵S-PTH-CBD (122 kcm/mcg)was injected subcutaneously in 7 week-old mice (32-35 g). Mice weresacrificed at 1 hour or 12 hours post-injection, and then frozen in dryice-acetone. Frozen sections (50 μm) were prepared with an autocryotome,dried at −20° C., and exposed to an image plate for 4 weeks. Thereappeared to be an initial distribution of ³⁵S-PTH-CBD to a broad area ofskin around the site of injection, followed by a rapid redistribution tothe skin of the entire animal, as well as to several other tissues (i.e.bone, intestine, bladder) (FIG. 2). PTH-CBD thus showed the desiredproperties of distribution and retention to skin with subcutaneousadministration.

PTH-CBD Reverses Hair Loss in Chemotherapy-Induced Alopecia in Mice:

We compared efficacy of CBD linked PTH agonists and antagonists inchemotherapy-induced alopecia, utilizing an experimental designpublished by Peters, et al., for non-CBD linked PTH compounds. Peters,et al. (2001) J Invest Dermatol 117:173-178. C57BL/6J mice (JacksonLaboratories, Bar Harbor, Me.) were depilated to synchronize the hairfollicles, and cyclophosphamide (CYP, 150 mg/kg) was administered on day9 to maximize the chemotherapy-induced damage. The agonist (PTH-CBD) andthe antagonist (PTH(7-33)-CBD) were administered 2 days prior tochemotherapy, and given the long-term retention of the compounds in theskin, we administered only a single dose to cover the timing of themultiple injections of PTH agonist and antagonist in the study byPeters, et. al. The administered dose of CBD-linked compounds (320meg/kg) is well tolerated in mice. Ponnapakkam et al. (2011) Calcif88511-520. Epub 2011 April 2022.

The results of the photodocumentation record indicate that the agonist,PTH-CBD, was far more effective at stimulating hair growth than was theantagonist (FIG. 3). Histological examination revealed morphologicalchanges in the hair follicles after CYP therapy, which were moresuperficially located and exhibited clumped melanocytes around the bulb,characteristics of the dystrophic anagen and catagen phase (FIG. 4).While the antagonist PTH(7-33)-CBD had no beneficial effect, treatmentwith the agonist PTH-CBD led to deeper rooting and reduced melanocyteclumping, thus reversing the dystrophic changes. Counts of anagen VIhair follicles per high-powered field (HPF) were compared betweengroups; animals treated with PTH-CBD had a higher number of hairfollicles, approaching those of animals which did not receivechemotherapy (FIG. 5), while the antagonist PTH(7-33)-CBD had nobeneficial effect.

Importantly, we saw no evidence of adverse effects from PTH-CBDadministration. While PTH injections are known to elevate blood calciumand can cause kidney stones, PTH-CBD had no effect on serum calcium. Inaddition, there was no evidence of excess hair length on the body or ofexcess hair growth on the ears and tail, where a full coat is normallynot present. The effects of PTH-CBD on hair growth have been confirmedin models of chemotherapy-induced alopecia without depilation, whichmore closely mimic clinical protocols.

Quantification of Effects of PTH-CBD in Chemotherapy-Induced Alopecia:

We followed these studies by comparing the effects of different doses ofPTH-CBD in chemotherapy-induced alopecia. In these studies, we appliedthe injections more distally on the back and applied a gray-scaleanalysis to quantify the amount of hair growth. Injecting more distallyin the back allows us to compare regrowth of hair after PTH-CBDtreatment with less interference from the normal hair regrowth, whichnormally proceeds from head to tail in mice. The results are shown inFIG. 6, indicating a dose-dependent effect on hair regrowth bothqualitatively and quantitatively.

Chemotherapy-Induced Alopecia without Depilation:

While the depilated model of chemotherapy-induced alopecia provides auniform model for comparison of drug effects, the depilation process isknown to cause hair follicle injury, and may alter the response of theanimals to the PTH-CBD administration. We therefore tested the effectsof PTH-CBD in another model of chemotherapy-induced alopecia, where theanimals were given 3 courses of cyclophosphamide therapy (50 mg/kg/wk),similar to the usual manner in which cancer patients might be treated.In this model, it takes much longer (4-6 months) for alopecia todevelop. Animals that received a single dose of PTH-CBD (320 meg/kgsubcutaneous) prior to the first cycle did not develop hair loss asshown in FIG. 7.

In a second study, we compared the effects of PTH-CBD when givenprophylactically, at the time of the first cycle of chemotherapy, vs.therapeutically, after the hair loss had developed. While PTH-CBD waseffective in both instances, the effects were more prominent when givenprophylactically. This is evident both visually and quantitatively inFIG. 8, using the same grey scale analysis used in our dose-responsestudy.

Depilation Alopecia:

The agonist PTH-CBD appears to increase hair growth by increasing thenumber of anagen phase hair follicles. As such, there is no reason tobelieve that hair growth effects should be limited to the chemotherapymodel. We therefore tested both PTH-CBD and antagonist compound,PTH(7-33)-CBD, after removing hair from C57/BL6J mice by waxing (FIG.9). The results were quite interesting; agonist (PTH-CBD) treatedanimals had earlier anagen eruption (day 7 vs. day 9 for vehiclecontrols), and exhibited more complete regrowth of hair by the end ofthe study (day 18). Antagonist (PTH(7-33)-CBD) treated animals also hadan early anagen eruption, but the hair growth which followed wasmarkedly curtailed, and the hair cycle was arrested after this point,resulting no further observed regrowth of hair. Thus, it appears thatagonist therapy is acting to promote more rapid regrowth of hair bypromoting more rapid transition to the anagen phase, while theantagonist inhibited hair regrowth by blocking this transition.

PTH-CBD is a fusion protein of the first 33 amino acids of parathyroidhormone (PTH) and a bacterial collagen binding domain. The collagenbinding activity causes PTH-CBD to be retained at its site of action inthe dermal collagen, maximizing efficacy and reducing systemicside-effects. PTH-CBD stimulates hair growth by causing hair folliclesto enter an anagen VI or growth phase, presumably by activating WNTsignaling and increasing production of beta-catenin. We therefore planto conduct the following additional studies to confirm this mechanism ofaction and to determine the effect of PTH-CBD in two distinct geneticmouse models with WNT signaling inhibition. These data will be used informulating clinical trials for PTH-CBD as a therapy for alopecia.

Alopecia Areata Alopecia:

Areata is a disease of patchy hair loss due to autoimmune destruction ofthe hair follicles. We tested the efficacy of PTH-CBD in promotingregrowth of hair in an animal model of alopecia areata, the engraftedC3H/Hej mouse. In this model, hair loss develops variably over the first2 months of life. Shown in FIG. 10 is the results of a single dose ofPTH-CBD (320 meg/kg subcutaneous) administered into the engrafted site,the center of the back, where there was maximal hair loss. Compared tovehicle control animals, which continued to lose hair at this site,animals receiving PTH-CBD began to show regrowth of hair within the next1-4 days. Importantly, the response was found to be sustained during the2 month course of the experiment.

Example 3 CBD-PTH can Prevent or Treat Hyperparathyroidism

In this experiment, rats had their ovaries surgically removed at age 3months. At age 9 months, rats were injected with either a single dose ofPTH-CBD (320 mcg/kg) or vehicle control. Animals were sacrificed 6months after therapy (age 15 months). Human intact PTH levels weremeasured to assess serum levels of PTH-CBD, and were found to beundetectable in both groups. Serum calcium was measured and there wereno differences between groups (Vehicle: 13.5+/−1.1 vs. PTH-CBD:14.3+/−1.1 mg/dl, NS). Rat intact PTH levels were measured to assessendogenous PTH production, and PTH-CBD suppressed the normal increase inendogenous PTH levels seen in aged, ovarectomized rats (FIG. 11). Thesefindings indicate that a single injection of PTH-CBD can providelong-term suppression of endogenous PTH production, preventing thenormal rise seen with age in the ovarectomized rat model, and thus mayserve as a therapy for hyperparathyroidism.

We claim:
 1. A method of slowing hair growth comprising administering acomposition comprising a bacterial collagen-binding polypeptide segmentlinked to a PTH/PTHrP receptor antagonist to a subject to slow hairgrowth relative to the hair growth the subject would experience withouttreatment, wherein the bacterial collagen-binding polypeptide segmentcomprises a collagen-binding polypeptide derived from an M9 peptidaseselected from the group consisting of, one of SEQ ID NOs: 13-34 or afragment of at least 8 consecutive amino acids of SEQ ID NOs: 13-34,residues 34-158 of SEQ ID NO: 1, a fragment of at least 8 consecutiveamino acids from residues 34-158 of SEQ ID NO: 1, and a peptide that isat least 90% identical to residues 34-158 of SEQ ID NO: 1 or SEQ ID NOs:13-34, and wherein the PTH/PTHrP receptor antagonist comprises asequence selected from the group consisting of residues 7-33 of SEQ IDNO: 7, residues 7-14 of SEQ ID NO: 7, a fragment of at least 8consecutive amino acids from residues 7-34 of SEQ ID NO: 7, and residues((−1)-33) of PTH.
 2. The method of claim 1, wherein the composition isadministered locally.
 3. The method of claim 2, wherein the compositionis administered topically.
 4. The method of claim 1, wherein and theN-terminus of the collagen-binding polypeptide segment is linkeddirectly or through a linker polypeptide segment to the C-terminus ofthe PTH/PTHrP receptor antagonist polypeptide.
 5. The method of claim 1,wherein the collagen-binding polypeptide segment and the PTH/PTHrPreceptor antagonist polypeptide are chemically cross-linked to eachother or are polypeptide portions of a fusion protein.
 6. The method ofclaim 1, wherein the subject is human.
 7. The method of claim 1, whereinthe bacterial collagen-binding polypeptide segment comprises residues34-158 of SEQ ID NO: 1 or a peptide that is at 90% identical to residues34-158 of SEQ ID NO:
 1. 8. The method of claim 1, wherein the PTH/PTHrPreceptor antagonist comprises residues 7-33 of SEQ ID NO:
 7. 9. Themethod of claim 1, wherein the bacterial collagen-binding polypeptidesegment linked to the PTH/PTHrP receptor antagonist is a fusion proteincomprising SEQ ID NO:
 10. 10. The method of claim 4, wherein the linkerpolypeptide segment comprises a PKD domain.
 11. The method of claim 10,wherein the PKD domain comprises residues 807-901 of SEQ ID NO:
 6. 12.The method of claim 1, wherein the composition is administered after thesubject undergoes a hair removal procedure.